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Cysteine protease enhances plant-mediated bollworm RNA interference.

Mao YB, Xue XY, Tao XY, Yang CQ, Wang LJ, Chen XY - Plant Mol. Biol. (2013)

Bottom Line: Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant.We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines.Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People's Republic of China.

ABSTRACT
Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

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The effects of cysteine protease on bollworm midgut PM permeability to gossypol. Varied gossypol accumulation in midgut after bollworm ingestion of cysteine proteases. The 3rd instar larvae were fed with artificial diet supplemented with E. coli expressing His-tag fusion proteins of GFP, GhCP1 or AtCP2, respectively (a), or with the purified fusion proteins (b) for 8 h, and then transferred to gossypol (1 mg/g)-supplemented diet for another 16 h; gossypol content in midgut was measured by a phloroglucinol/HCl assay. To 10 g artificial diet, precipitates of 50 ml liquid culture of E. coli cells (OD 1.0), or purified His-GhCP1, His-AtCP2 or His-GFP proteins (10 mg each), respectively, were added; Accumulation of gossypol in midgut cells of the bollworms that were pre-fed with 35S:GhCP1 (c) or 35S:AtCP2 (d) plant leaves. The 3rd instar larvae were fed on wild-type (WT) or transgenic Arabidopsis leaves for 2 days, then transferred to diet containing gossypol (1 mg/g) for another day. Data are shown as mean ± SD. * p < 0.05
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Fig3: The effects of cysteine protease on bollworm midgut PM permeability to gossypol. Varied gossypol accumulation in midgut after bollworm ingestion of cysteine proteases. The 3rd instar larvae were fed with artificial diet supplemented with E. coli expressing His-tag fusion proteins of GFP, GhCP1 or AtCP2, respectively (a), or with the purified fusion proteins (b) for 8 h, and then transferred to gossypol (1 mg/g)-supplemented diet for another 16 h; gossypol content in midgut was measured by a phloroglucinol/HCl assay. To 10 g artificial diet, precipitates of 50 ml liquid culture of E. coli cells (OD 1.0), or purified His-GhCP1, His-AtCP2 or His-GFP proteins (10 mg each), respectively, were added; Accumulation of gossypol in midgut cells of the bollworms that were pre-fed with 35S:GhCP1 (c) or 35S:AtCP2 (d) plant leaves. The 3rd instar larvae were fed on wild-type (WT) or transgenic Arabidopsis leaves for 2 days, then transferred to diet containing gossypol (1 mg/g) for another day. Data are shown as mean ± SD. * p < 0.05

Mentions: To examine the effect of cysteine proteases on midgut PM structure of cotton bollworm, the PM was isolated from 5th instar larvae and incubated with 2 mg/ml purified GFP, GhCP1 and AtCP2, respectively. After incubation, PM proteins were visualized by Coomassie blue staining. We found that the staining became much paler if the PMs were treated with GhCP1 or AtCP2 (Fig. S3), indicating that GhCP1 and AtCP2 could affect the PM structure by digesting the PM proteins. Next, we investigated whether ingested GhCP1 and AtCP2 would affect PM permeability. The 3rd instar larvae were fed with artificial diet supplemented with E. coli cells expressing His-GhCP1, His-AtCP2, or His-GFP, respectively. After 8 h, the larvae were transferred to artificial diet containing 1 mg/g gossypol. The amount of gossypol in midgut was measured after 16 h. We found that the larvae pre-fed with His-GFP E. coli accumulated 129 ng gossypol per larva in their midgut, whereas those with His-GhCP1 or His-AtCP2 E. coli accumulated 182 and 241 ng per larva, respectively, significantly higher than the control (Fig. 3a). Similar feeding assay results were obtained when the bacterial cells were replaced by the purified fusion proteins (Fig. 3b).Fig. 3


Cysteine protease enhances plant-mediated bollworm RNA interference.

Mao YB, Xue XY, Tao XY, Yang CQ, Wang LJ, Chen XY - Plant Mol. Biol. (2013)

The effects of cysteine protease on bollworm midgut PM permeability to gossypol. Varied gossypol accumulation in midgut after bollworm ingestion of cysteine proteases. The 3rd instar larvae were fed with artificial diet supplemented with E. coli expressing His-tag fusion proteins of GFP, GhCP1 or AtCP2, respectively (a), or with the purified fusion proteins (b) for 8 h, and then transferred to gossypol (1 mg/g)-supplemented diet for another 16 h; gossypol content in midgut was measured by a phloroglucinol/HCl assay. To 10 g artificial diet, precipitates of 50 ml liquid culture of E. coli cells (OD 1.0), or purified His-GhCP1, His-AtCP2 or His-GFP proteins (10 mg each), respectively, were added; Accumulation of gossypol in midgut cells of the bollworms that were pre-fed with 35S:GhCP1 (c) or 35S:AtCP2 (d) plant leaves. The 3rd instar larvae were fed on wild-type (WT) or transgenic Arabidopsis leaves for 2 days, then transferred to diet containing gossypol (1 mg/g) for another day. Data are shown as mean ± SD. * p < 0.05
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Related In: Results  -  Collection

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Fig3: The effects of cysteine protease on bollworm midgut PM permeability to gossypol. Varied gossypol accumulation in midgut after bollworm ingestion of cysteine proteases. The 3rd instar larvae were fed with artificial diet supplemented with E. coli expressing His-tag fusion proteins of GFP, GhCP1 or AtCP2, respectively (a), or with the purified fusion proteins (b) for 8 h, and then transferred to gossypol (1 mg/g)-supplemented diet for another 16 h; gossypol content in midgut was measured by a phloroglucinol/HCl assay. To 10 g artificial diet, precipitates of 50 ml liquid culture of E. coli cells (OD 1.0), or purified His-GhCP1, His-AtCP2 or His-GFP proteins (10 mg each), respectively, were added; Accumulation of gossypol in midgut cells of the bollworms that were pre-fed with 35S:GhCP1 (c) or 35S:AtCP2 (d) plant leaves. The 3rd instar larvae were fed on wild-type (WT) or transgenic Arabidopsis leaves for 2 days, then transferred to diet containing gossypol (1 mg/g) for another day. Data are shown as mean ± SD. * p < 0.05
Mentions: To examine the effect of cysteine proteases on midgut PM structure of cotton bollworm, the PM was isolated from 5th instar larvae and incubated with 2 mg/ml purified GFP, GhCP1 and AtCP2, respectively. After incubation, PM proteins were visualized by Coomassie blue staining. We found that the staining became much paler if the PMs were treated with GhCP1 or AtCP2 (Fig. S3), indicating that GhCP1 and AtCP2 could affect the PM structure by digesting the PM proteins. Next, we investigated whether ingested GhCP1 and AtCP2 would affect PM permeability. The 3rd instar larvae were fed with artificial diet supplemented with E. coli cells expressing His-GhCP1, His-AtCP2, or His-GFP, respectively. After 8 h, the larvae were transferred to artificial diet containing 1 mg/g gossypol. The amount of gossypol in midgut was measured after 16 h. We found that the larvae pre-fed with His-GFP E. coli accumulated 129 ng gossypol per larva in their midgut, whereas those with His-GhCP1 or His-AtCP2 E. coli accumulated 182 and 241 ng per larva, respectively, significantly higher than the control (Fig. 3a). Similar feeding assay results were obtained when the bacterial cells were replaced by the purified fusion proteins (Fig. 3b).Fig. 3

Bottom Line: Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant.We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines.Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People's Republic of China.

ABSTRACT
Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

Show MeSH
Related in: MedlinePlus